Interactomics: Protein Arrays and Label-Free Biosensors.

Professor Sanjeeva Srivastava.

Department of Biosciences and Bioengineering.
Indian Institute of Technology, Bombay.
Lecture-8.
Protein-Protein Interaction Study: Binding Analysis.
Welcome to the MOOC NPTEL course on interactomics. To completely understand the
function of a protein, it is important to look beyond its expression pattern and identify its
potential interaction partners and determine their interaction dynamics. To study the
interaction between two binding partners in SPR experiment, one partner is attached to the
surface and other is passed over the surface in a continuous flow of sample solution.

The interaction of ligand and analyte is measured by the SPR instrument as a change in the
refractive index over time and the response observed is directly proportional to the change in
mass concentration close to the surface. In the previous lecture, we completed immobilization
procedure of anti-beta-2 microglobulin antibody on the surface of a CM5 chip. We will now
move forward to perform the binding analysis of anti-beta-2 microglobulin antibody with
beta-2 microglobulin protein.

(Refer Slide Time: 1:51)

So, let us have the experimental session now. We will go into the second session now and we
will use our immobilized chip for a binding experiment. So, here we will first prepare a
template for the binding experiment. A binding experiment actually deals with screening or a
single concentration screen of different compounds on the immobilized ligand and look for
its ability to interact with the target that is immobilized on the surface.
(Refer Slide Time: 2:41)

Before we go ahead with the binding experiment, let us understand some important
considerations, during sample injection the analyte is injected over the surface with a constant
flow and concentration. Analyte in the sample binds to the immobilized ligand on the surface,
the mass on the surface changes and the response is recorded. After sample injection buffer
flows over the surface to allow monitoring of analyte dissociation from the ligand.

Regeneration as already discussed by Dr. Srinivas, is the process of removing bound analyte
from the ligand on the sensor chip surface after analysis of a sample. Efficient regeneration
which means removing bound analyte without affecting the ligand activity is crucial to a
successful assay. If the regeneration is incomplete or the binding activity of the surface is
reduced, the performance of the assay is impaired.
(Refer Slide Time: 4:10)

The choice of conditions for regeneration is dictated by the stability and nature of the ligand
and analyte. In today’s binding experimental setup, we will be using HEPES-EP plus as the
running buffer, we will be preparing three different concentrations of beta-2 microglobulin
protein which are 8.5 nanomolar, 42.5 nanomolar and 85 nanomolar for evaluating its binding
with the antibody having 8.5 nanomolar concentration in duplicate. These three
concentrations will be referred to as low, medium and high.

Contact time between the sample and the sensor surface should be sufficient to give
confidently measurable response levels without compromising screening throughput. Contact
time of 1 to 2 minutes are usually sufficient for a binding experiment. Here, we will provide a
contact time of 60 seconds at the flow rate of 10 microliter per minute with the dissociation
time of 60 seconds. An ideal regeneration condition is the one where analyte response of the
same concentration is constant after repeated injections.
(Refer Slide Time: 5:53)
Today, we will be using 10 milimolar glycine pH 2.5 for regeneration of the surface; we will
now proceed with our binding experiment protocol. Before making the template we will open
the file wizard again, binding analysis new identify the flow path as 2 minus 1 as we have
done our immobilization on 2 minus 1, chip will be CM5 that is docked already, we will not
have ligand capture, sample and regeneration.

We will go to the next tab, here we are not using any conditioning cycle, we will start with
the startup cycle. Startup cycles are cycles of buffer used for equilibrating the system. So,
here basically buffer is used as analyte. So, we can type as HEPES-EP plus buffer and from
the pull down menu, we will select 3 cycles. Generally for binding experiments, 3 cycles are
selected, going to the next tab for setting up of binding, we need to specify the contact time as
60 seconds default flow rate of 10 microliter per minutes. Dissociation time of any minute or
any second by default we can consider 60 seconds.

The regeneration solution we would prefer here would be 10 milimolar glycine pH 2.5 with
the default contact time of 30 seconds, flow rate of 30 microliter per minute and with no
stabilization time. We go to the next tab; here we need to fill in the name of all single
concentration compounds. So, here we would select our analyte as beta-2M, just that we have
one analyte, we will take it in three different concentrations. So, we will name as low beta-
2M, medium and beta-2M high.

So, low indicates lower concentration, medium concentration, high concentration and we will
go to the next tab we will collect prime before run and normalize is not required here because
the chip is already immobilized and we will go with the default temperatures and we will go
to the next tab here, we will not select a micro-titer plate and this is our rack positions for a
binding experiment. Here, we have at the C1 position HEPES-EP plus buffer for three
different startups. We have three concentrations of analyte; high, medium and low and we
have the regeneration solution here which is 10 milimolar glycine pH 2.5 and we will prepare
our solutions and start the binding experiment.

(Refer Slide Time: 11:12)

We will now work on the reagent require for the binding analysis of anti-beta-2
microglobulin with beta-2 microglobulin proteins. We will be using HEPES-EP plus as the
running buffer which will also be used for the initial startup cycles. We will tell you the stock
solution of protein that is 100 microgram per ml in the running buffer HEPES-EP plus to
prepare 100 microliter of three different concentrations that is 85 nanomolar, 42.5 nanomolar
and 8.5 nanomolar which are referred to as high, medium and low concentrations in the
experiment.

We will also include one zero nanomolar concentration in the experiment which will be
nothing but the running buffer. For the regeneration of the surface we have prepared glycine
HCl pH 2.5 as the regeneration solution, we have transferred all the solution in the
specialized tube starting from the startup, beta-2M concentrations starting from 85
nanomolar, 42.5 nanomolar, 8.5 nanomolar and zero nanomolar.
(Refer Slide Time: 13:34)
The regeneration solution is placed in this glass vial. We will now insert these tubes into the
appropriate rack and then into this system to start with the binding analysis of anti-beta-2
microglobulin with beta-2 microglobulin protein. We will eject the rack now, to insert new
vials, eject rack and take the plate out of the sample rack and we will fill it with the binding
vials.

So, the vials positions filled with different samples as you can see on this screen, the startup
is here. So, beta-2 M medium, beta-2 M low, beta-2 M high are put at their respective
positions and vial for regeneration of 10 milimolar glycine pH 2.5 is here. Now, we close the
rack and will be inserted in the sample compartment by ejecting the rack compartment,
inserting the plate next tab again we need to do all these checks, check the prime.

The estimated run time of 38 minutes and we have sufficient amount of buffer and we will
now, start the experiment; we will save this template as binding save. Now, we will save the
result vial again as binding. And now, the experiment has started, shows running binding
analysis with an estimated time of 38 minutes. System is priming now and once we finish off
the binding experiment we will take a look at the data.
(Refer Slide Time: 17:47)
Before we analyse the binding data from the experiment, let us look at a typical sensorgram
for binding between a ligand and an analyte. A sensorgram as shown here is a plot of
response against time showing the progress of interaction. This curve is displayed on the
system during the course of experiment, we observe the base line followed by the injection of
analyte which leads to increase in the binding response during the association phase.

Just after this top of the sample injection, we observe report point which records the response
on a sensorgram at a specific time averaged over a short time window. This is followed by
dissociation phase regeneration and then back to baseline.

(Refer Slide Time: 19:37)

We will now proceed to analyse the data obtained from binding of anti-beta-2 microglobulin
with beta-2 microglobulin protein, after finish of our binding experiment by double click on
the file, the file is open now, you can see here from our binding experiment, it shows all
sensorgram here. So, here the green ones are our startups. So, we have setup 3 or 5 different
startups and the red ones are our actual data from beta-2 microglobulin. What we will do is
we will highlight only our sampled data.

And now, you see the data for beta-2 M. We go on to sensorgram adjustment to report point
on the vial adjustment baseline and save ok. Now our data is baseline to zero if we want we
can as well go on to color sample and we will see the different samples in different color with
the ligand on one side of the screen. Here, we have low, medium and high concentrations of
beta-2 microglobulin injected over anti-beta-2 M; we could subtract or delete the regeneration
area cut. Now, we can see our different concentrations of beta-2 M with one of them in
duplicate.

So, there is definitely binding of the beta-2 M to anti-beta-2 M antibody in a dose dependent
manner. The data can also be shown in the form of a bar chart with all our red startup runs
and green as our sample runs. Here, we will highlight the sample runs only and see cycle 7, 8
and 9 are our low, medium and high concentration data which are dose dependent binding.

Going to another tab we can see the relative responses of each molecule from the binding at 4
for the duplicates and medium at 29 and high at 52. With this we will conclude our binding
session and we prepare now for a kinetics experiment.

(Refer Slide Time: 24:40)

Protein interactions are identified using a wide array of applications however what is also
required is an understanding of the extent to which these interactions actually occur.
Therefore, performing protein-protein interaction studies and calculating their kinetic values
becomes very crucial. Let us continue our discussion and SPR experiment for the kinetics
analysis in next lecture. Thank you.

(Refer Slide Time: 25:31)